cDNA from hepatitis A virus (HAV) has been cloned into pBR322. Six cDNA clones which together span the entire genome were isolated and ligated together to form a single clone thought to represent full length HAV cDNA. Transfection of both tissue culture cells (in vitro) and marmosets (in vivo) with these plasmids failed to generate HAV. Fine structure mapping of the HAV cDNA indicated that about 40 base pairs had been deleted during the ligation process. The deletion was repaired, but transfection of marmosets and tissue culture cells still failed to generate HAV. An additional modified construct, differing by two nucleotides thought to be important for infectivity, also failed to generate HAV in marmosets. The entire full length construct was sequenced; comparison with the sequence of the parent clones used to create the construct did not reveal any differences. The cDNA was placed into an RNA transcription vector and plus strand RNA was made in vitro from the cDNA. Transfection of marmosets with the RNA failed to generate HAV. Preparation of plus strand HAV RNA, synthesized in vitro from minus strand HAV RNA and poliovirus RNA polymerase, is in progress.